The Nef gene of HIV-1 encodes a small dimeric protein essential for AIDS progression. Despite intensive investigation of Nef structure and function, few attempts have been made to target Nef or its downstream signaling partners with small molecule inhibitors. This application proposes novel high-throughput screens (HTS) to identify inhibitors of Nef signaling complexes. One major focus will be the protein-tyrosine I-Ick, a member of the Src kinase family selectively expressed in macrophages. Nef binding induces constitutive Hck kinase activation and contributes to survival signaling in macrophages, an HIV target cell and viral reservoir. In addition, genetic studies support a requirement for Hck in a mouse model of Nef-induced AIDS progression. Discovery of small molecule inhibitors of Nef signaling will involve complementary in vitro and cell-based approaches: Aim 1: Test the hypothesis that the Hck-Nef complex represents a unique target for selective inhibitor discovery. This Aim proposes a screen for selective inhibitors of the Hck-Nef complex in vitro. Use of the Hck-Nef complex as the source of active kinase in the assay will provide a rich source of molecular targets for inhibitor binding. Aim 2: Develop a yeast-based screen for conformationally selective inhibitors of the Hck-Nef complex. This approach will identify compounds that selectively bind to the inactive form of Hck prior to interaction with Nef. Aim 3: Develop a cell-based screen to identify inhibitors of Nef survival signaling in an HIV target cell lineage. This system will use an inducible form of Nef to identify selective inhibitors of Nef-dependent proliferation and survival signaling in a macrophage precursor cell line. Aim 4: Develop phosphoproteomics approaches to define the tyrosine kinase signaling networks affected by Nef in myeloid cells and identify mechanisms of inhibitor action in vivo. This Aim will employ high-resolution 2D gels, anti-phosphotyrosine immunoblotting and mass spectrometry to produce a global picture of Nef-tyrosine kinase signaling in macrophage precursor cells, providing a powerful tool to define the molecular targets for newly discovered small molecule inhibitors. Identification of a selective inhibitor of Hck-Nef signaling will provide an invaluable tool to test the role of this protein complex in AIDS progression as well as an important lead for future drug discovery.